Interaction with intracellular components of a cell requires that the cellular membrane is crossed by an agent that is expected to interact with such intracellular components. However, such agents often lack the necessary balance of biological and physicochemical properties such as hydrophobicity, solubility, charge and size to cross the cell membrane.
In the therapeutic field, macromolecules (such as polypeptides and nucleic acids) have been the main focus because for many diseases, small molecule drugs (i.e. chemical compounds containing less than 100 atoms) are very difficult to find and/or develop. The use of macromolecules as therapeutic agents has a number of advantages over small molecules, the most important one being the ability to adopt large, stable three-dimensional conformations suitable for strong binding to targets, thereby allowing to interfere with native protein-protein or protein-nucleic acid interfaces that are difficult to address using small molecules. Moreover, the stability, size, and complexity of macromolecules can result in specificities that are not easily achievable using small molecules.
However, a great difficulty is that macromolecules as such are not able to diffuse into cells, and thus, while the great majority of disease targets of interest are located inside cells, most macromolecule therapeutics are only capable of addressing extracellular targets. Accordingly, over the years, various approaches for intracellular delivery of macromolecules have been developed, including electroporation, ultrasound-mediated plasmid delivery, viral delivery, nebulization, and direct chemical modification. Other strategies associate a macromolecule with a non-viral delivery vehicle such as lipidoids, liposomes, dendrimers, cationic polymers, inorganic nanoparticles, carbon nanotubes, cell-penetrating peptides, small molecules, or receptor ligands. The use of supercharged proteins as vectors or carriers for macromolecule delivery into mammalian cells has been reported (Lawrence, M. S. et al. 2007, J. Am. Chem. Soc. 129, (33), 10110-10112; Cronican et al. 2010 ACS CHEMICAL BIOLOGY; McNaughton et al. 2009 PNAS).
Although these strategies may facilitate internalization, their applicability is restricted. For example, these methods utilize cellular mechanisms of internalization leading to accumulation of an effector in the lysosomes and ultimately resulting in degradation and inactivation of the effector compound.